Molecular Cloning And Expressing Analysis Of Cadm2b In Adult Tissues Of Grass Crap,Ctenopharyngodon Idellus

CADMs (cell adhesion molecules) family are vertebrate-specific multifunctional cell adhesion molecules. They play important roles in establishing the first line of defense against illnesses and infections by mediating adhesion between immune mast cells and their target cells. Grass carp (Ctenopharyngodon idellus) have a low livability at the age of 1、2 due to its susceptibility to infection cause by infectious virus or bacterium. To investigate whether CADMs participate in building the first line of defense against infection in grass carp, we cloned the mRNA of grass carp cadm2b. Using RT-PCR, RACE method and sequencing analysis, we identify 4 different full length cDNA of cadm2b from grass carp brain tissue. According to sequence alignment, sequences of the 5’terminal are identical among all the four cDNAs while various deleted fragments can be found in three different position in their 3’terminals. This observation indicates that all the four mRNAs, named cadm2b, cadm2b X2, cadm2b X3 and cadm2b X6, are splicing variants transcribed from cadm2b gene. The full length of cadm2b is 1669 bp, with a 1203bp long open reading frame(ORF) which codes 400 amino acids. The full length of cadm2b X2 is 2783 bp, with a 1323bp long ORF which codes 440 amino acids. The full length of cadm2b X3 is 2755bp, with a 1296bp long ORF which codes 431 amino acids. The full length of cadm2b X6 cDNA is 2649bp, with a 1161 bp long ORF which codes 386 amino acids. Prediction of amino acid sequence and protein sequence base on nucleotide sequence showed that all the four CADM2b isoforms contain the four conserve functional domains of CADM family in the N-terminals, but the C-terminas are variant. CADM2b has a juxtamembrane 4.1 protein binding domain and PDZ type Ⅱ protein binding domain in the C-terminal. Both CADM2b X2 and CADM2b X3 lacks the PDZ type Ⅱ protein binding domain. CADM2b X6 possesses neither the juxtamembrane 4.1 protein binding domain nor the PDZ type Ⅱ protein binding domain. Quantitative RT-PCR analysis suggested that splicing variant cadm2b might be the main form of cadm2b mRNA. Expression pattern of cadm2b in adult tissues of grass carp were examined through semi-quantitative RT-PCR and nested primer PCRexperiments. A high-level expression was detected in brain tissue and a very low-level expression was detected in liver, kidney, heart and muscle. This expression pattern suggests that CADM2b is a cell adhesion molecules synthesized and secreted by nonimmune cells, rather than by immune mast cells, and might play a role in against various infections by mediating adhesion between immune mast cells and their target cells in grass carp.